Altered signaling by neuromodulators, such as biogenic amines or neuropeptides, underlies human brain diseases including mood disorders and Parkinson's disease, yet our understanding of exactly how neuromodulators produce proper functioning of neural circuit's remains vague. How do neuromodulators collaborate with fast-acting neurotransmitters to create patterns of activity in neural circuits that underlie complex behaviors? Our goal is to understand for the first time precisely how neuromodulators function in a model neural circuit. The egg-laying circuit of C. elegans contains just three types of neurons and one set of postsynaptic muscles. This circuit involves just one fast-acting neurotransmitter, acetylcholine, plus a set of neuromodulators: the biogenic amines serotonin and tyramine, and several neuropeptides. The circuit generates a two-state behavior that alternates between a ~2.5 minute active phase in which rhythmic egg-laying contractions occur, and a ~20 minute inactive phase. Over the past 16 years, we have developed a battery of C. elegans mutants that show increased or decreased egg laying due to defects in specific signaling molecules in the circuit. We have also developed cell-specific promoters that allow us to express any protein we want in any individual cell type of the egg-laying system. We have now developed new tools to optogenetically manipulate activity of the individual cell types and to use the genetically-encoded Ca2+ sensor GCaMP to quantitatively analyze activity of each cell type within freely-behaving animals. These new tools have brought us to a tipping point at which we are ready to delineate how each cell and each neurotransmitter in the circuit act to together produce the activity pattern of the circuit. We expect a newly meaningful understanding of neuromodulators will be revealed now that we can finally analyze their function in the context of detailed investigation of a circuit they modulate. We will generate this understanding through three aims, each focused on dissecting the function one of the three neuron types in the circuit: Aim 1. We will determine how serotonin and the neuropeptide NLP-3, co-released from the HSN motor neuron, signal onto other cells in the circuit to initiate the active phase of egg laying. Aim 2. W will determine how acetylcholine, released from the VC motor neurons within the active phase, acutely triggers egg-laying contractions only when serotonin and NLP-3 have activated the circuit. Aim 3. We will determine how the uv1 neuroendocrine cells release tyramine to lengthen the inactive phase of egg laying and to space out egg-laying events during the active phase.